Nucleic acid amplification method

a technology of nucleic acid and amplification method, which is applied in the direction of microbiological testing/measurement, biochemistry apparatus and processes, fermentation, etc., can solve the problems of difficult primer design, complicated temperature control, and increased time loss in proportion to the number

Active Publication Date: 2009-07-02
FUJIFILM CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, implementation of nucleic acid amplification reactions at three different types of temperature is problematic in that temperature control is complicated and time loss increases in proportion to the number of cycles.
However, the use of exonuclease in addition to polymerase is required, and thus the method is expensive and the design of primers should be improved.
However, the method requires the use of at least four types of primer that recognize six specific sites, so that the design of primers is very difficult.
However, this method also requires the use of special primers that are chimeric primers and thus the design of such primers is very difficult.
However, the method disclosed in JP Patent Publication (Kohyo) No. 11-509406 A is problematic in that it requires a relatively long reaction time, for example.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Amplification of Target Nucleic Acid Sequence in Human Gene

(1) Preparation of Nucleic Acid Specimen Solution Containing Target Nucleic Acid Fragment

[0115]7.5 ng of Human Genomic DNA (produced by Clontech) was heated at 98° C. for 3 minutes, and then a specific sequence in the target nucleic acid was amplified under the following conditions. As a negative control, a sample was also prepared by heating water under the same conditions.

[0116]The following 4 types of primers were designed, and purchased from Operon Biotechnologies. Each primer sequence is as shown below. Primers (1) and (2) are sequences of β-actin, and Primers (3) and (4) are complementary to sequences of β 2AR gene.

Primer (1):5′-GGGCATGGGTCAGAAGGATT-3′(SEQ ID NO: 1)Primer (2):5′-CCTCGTCGCCCACATAG-3′(SEQ ID NO: 2)Primer (3):5′-CTTGCTGGCACCCAATA-3′(SEQ ID NO: 3)Primer(4):5′-CCGGCGCATGGCTT-3′(SEQ ID NO: 4)

[0117]Tween 20 (Wako Pure Chemical Industries, Ltd.) was used as a surfactant. Tween 20 is polyoxyethylene(20) sorbita...

example 2

Effect of Concentration of the Surfactant

(1) Preparation of Nucleic Acid Specimen Solution Containing Target Nucleic Acid Fragment

[0139]7.5 ng of HumanGenomic DNA (produced by Clontech) was heated at 98° C. for 3 minutes, and then a specific sequence in the target nucleic acid was amplified under the following conditions. As a negative control, a sample was also prepared by heating water under the same conditions.

[0140]Primers (1) and (2) used in Example 1 were used as the primer.

Primer (1):5′-GGGCATGGGTCAGAAGGATT-3′(SEQ ID NO: 1)Primer (2):5′-CCTCGTCGCCCACATAG-3′(SEQ ID NO: 2)

[0141]Tween 20 (Wako Pure Chemical Industries, Ltd.) was used as a surfactant.

(2) Nucleic Acid Amplification Reaction

[0142]The amplification reaction was performed with the composition of a reaction solution shown below at 60° C. for 60 minutes. Bst. DNA polymerase (NEB (New England Biolabs)) was used as an enzyme.

[0143]

10 × Bst Buffer (Detergent Free)2.5 μL100 mM MgSO41.5 μL0% (v / v)-10% (v / v) Tween 201.25 μL ...

example 3

Effect of Type of the Surfactant

(1) Preparation of Nucleic Acid Specimen Solution Containing Target Nucleic Acid Fragment

[0149]7.5 ng of HumanGenomic DNA (produced by Clontech) was heated at 98° C. for 3 minutes, and then a specific sequence in the target nucleic acid was amplified under the following conditions. As a negative control, a sample was also prepared by heating water under the same conditions.

[0150]Primers (1) and (2) used in Examples 1 and 2 were used as the primer.

Primer (1):5′-GGGCATGGGTCAGAAGGATT-3′(SEQ ID NO: 1)Primer (2):5′-CCTCGTCGCCCACATAG-3′(SEQ ID NO: 2)

[0151]The following 9 types of substances were used as a surfactant, and experiment was carried out.

Level 1: Tween 40

[0152]Tween 40 is polyoxyethylene(20) sorbitan monopalmitate, and is a polyoxyethylene sorbitan fatty acid ester-based non-ionic surfactant. More particularly, Tween 40 is polyoxyethylene sorbitan fatty acid monoester. Tween 40 has HLB of 15.6, and is represented by the following formula. Tween 40...

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Abstract

An object to be achieved by the present invention is to provide a nucleic acid amplification method by which a nucleic acid can be amplified substantially isothermally using oligonucleotide primers and DNA polymerase capable of strand displacement. The present invention provides a nucleic acid amplification method which comprises performing substantially isothermal incubation of a reaction solution containing at least one type of deoxynucleotide triphosphate, at least one type of DNA polymerase having strand displacement activity, a divalent cation, at least 0.01% or more surfactant, at least two types of oligonucleotide primer, and the nucleic acid fragment as a template so as to perform a polymerase reaction that initiates from the 3′ end of the primer and thus amplifying the nucleic acid fragment.

Description

TECHNICAL FIELD[0001]The present invention relates to a nucleic acid amplification method. More specifically, the present invention relates to a nucleic acid amplification method that comprises performing a polymerase reaction through substantially isothermal incubation of a reaction solution using DNA polymerase capable of strand displacement.BACKGROUND ART[0002]In molecular biological research, nucleic acid amplification is generally performed by an enzymatic method using DNA polymerase. Polymerase chain reaction (PCR) is broadly known as a nucleic acid amplification method. For amplification of a target nucleic acid sequence, the PCR method comprises the three steps of: denaturing (denaturation step) double-stranded DNA as a template into single-stranded DNAs; annealing (annealing step) primers to the single-stranded DNAs; and elongating (elongation step) complementary strands using the primers as origins. According to a general PCR method, the denaturation step, the annealing st...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12P19/34
CPCC12Q1/6853C12Q2531/119C12Q2527/125C12Q2521/101
Inventor MIYOSHI, HAYATOIWAKI, YOSHIHIDEMORI, TOSHIHIRO
Owner FUJIFILM CORP
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